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Oxygen activation at the plasma membrane: relation between superoxide and hydroxyl radical production by isolated membranes

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Abstract

Production of reactive oxygen species (hydroxyl radicals, superoxide radicals and hydrogen peroxide) was studied using EPR spin-trapping techniques and specific dyes in isolated plasma membranes from the growing and the non-growing zones of hypocotyls and roots of etiolated soybean seedlings as well as coleoptiles and roots of etiolated maize seedlings. NAD(P)H mediated the production of superoxide in all plasma membrane samples. Hydroxyl radicals were only produced by the membranes of the hypocotyl growing zone when a Fenton catalyst (FeEDTA) was present. By contrast, in membranes from other parts of the seedlings a low rate of spontaneous hydroxyl radical formation was observed due to the presence of small amounts of tightly bound peroxidase. It is concluded that apoplastic hydroxyl radical generation depends fully, or for the most part, on peroxidase localized in the cell wall. In soybean plasma membranes from the growing zone of the hypocotyl pharmacological tests showed that the superoxide production could potentially be attributed to the action of at least two enzymes, an NADPH oxidase and, in the presence of menadione, a quinone reductase.

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Abbreviations

DEPMPO:

5-Diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide

DPI:

Diphenyleneiodonium

EPR:

Electron paramagnetic resonance

MD:

Menadione

NBT:

Nitro blue tetrazolium chloride

NQR:

Naphthoquinone reductase

POBN:

α-(4-Pyridyl-1-oxide)-N-tert-butylnitrone

RBOH:

Respiratory burst oxidase homologue

SOD:

Superoxide dismutase

XTT:

Na,3′-1-[phenylamino-carbonyl]-3,4-tetrazolium-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate

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Acknowledgments

We are grateful to Drs. F. Sparla and P. Trost (Università di Bologna, Italy) for stimulating discussions and provision of the NQR antibodies. Research costs were funded by CEA and CNRS.

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Correspondence to Anja Krieger-Liszkay.

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Heyno, E., Mary, V., Schopfer, P. et al. Oxygen activation at the plasma membrane: relation between superoxide and hydroxyl radical production by isolated membranes. Planta 234, 35–45 (2011). https://doi.org/10.1007/s00425-011-1379-y

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